Protein deglycosylation using Endoglycosidase F1. A.) Our protein of interest (2 ug) was incubated with endoglycosidase F1 and deglycosylation assessed by Western blotting. The generation of two unique bands represent protein species that have lost either one or two glycosylation modifications. B.) As a positive control, ribonuclease B was incubated with endo F1 and deglycosylation assessed by silver staining an SDS-PAGE gel. This protein is known to contain N-linked glycosylations, which are efficiently removed as seen by a decrease in band molecular weight.
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The Native Protein Deglycosylation Kit is used to remove N-linked oligosaccharides from glycoproteins under native conditions as opposed to more traditional methods. Glycosylation protein modifications are often located at less accessible positions within a folded protein and cannot be removed with the more standard PNGase F treatment without denaturation. If your downstream application requires a folded protein after deglycosylation (e.g., enzymatic or protein interaction assays), this kit may be required. This kit contains purified endoglycosidases F1, F2, and F3, as well as corresponding reaction buffers. These endoglycosidases have varying linkage specificities. F1 is able to remove oligomannose and hybrid oligosaccharide structures, while F2 and F3 remove biantennary and triantennary structures, respectively.
We want to determine the biological activity of our protein of interest after deglycosylation. Removal of glycosylations can be assessed by Western blot (decrease in molecular weight of protein band).
Our recombinant protein of interest is a protein known to contain N-linked glycosylations. This protein was produced in HEK 293 cells and purified by affinity chromatography utilizing its N-terminal FLAG-tag.
Our protein of interest (2 ug) was incubated with 0.02 units of endoglycosidase F1, F2, or F3 and the corresponding enzyme buffer for 4 hours at 37°C. The extent of deglycosylation was assessed by standard SDS-PAGE and Western blotting. Note: Reaction conditions may need to be altered for different protein substrates.
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As expected, our protein was deglycosylated with results always being very consistent between samples. Of interest, endoglycosidase F1 was active against our protein, but F2 and F3 had no effect. Knowing which enzyme deglycosylates your protein will tell you something about the nature of its oligosaccharide linkages.
We had previously tried using PNGase (Sigma, #G5166), which was unable to deglycosylate our protein of interest.
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